Purification procedures determine the proteasome activation properties of REGγ (PA28γ)

The proteasome activation properties of recombinant REGγ molecules depend on purification procedures. Prior to ammonium sulfate precipitation recombinant REGγ activates the trypsin-like catalytic subunit of the proteasome; afterwards it activates all three catalytic subunits. The expanded activation specificity is accompanied by reduced stability of the REGγ heptamer providing support for the idea that a “tight” REGγ heptamer suppresses the proteasome’s chymotrypsin-like and postglutamyl-preferring active sites. In an attempt to determine whether REGγ synthesized in mammalian cells also exhibits restricted activation properties, extracts were prepared from several mammalian organs and cell lines. Surprisingly, endogenous REGγ was found to be largely monomeric. In an alternate approach, COS7 cells were cotransfected with plasmids expressing FLAG-REGγ and REGγ. The expressed FLAG-REGγ molecules were shown to form oligomers with untagged REGγ subunits, and the mixed oligomers preferentially activated the proteasome’s trypsin-like subunit. Thus, REGγ molecules synthesized in mammalian cells also exhibit restricted activation properties.